The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants plus those of S. pombe to elucidate the physiological functions of the polyamines, and, in particular, to ascertain the physiological effects of polyamine deprivation. During the current year we have extended our work on the role of polyamines in protecting cells from the toxic effects of oxygen and hydrogen peroxide, using the various polyamine-deficient mutants of S. cerevisiae that we had constructed. We have demonstrated by fluorescence microscopy and spectofluorometric methods that polyamine-deficient cells accumulate intracellular reactive oxygen species (ROS), resulting in cell death eventually even in air. More ROS is found when the cells are grown in oxygen, presumably accounting for the increased death of these cells in oxygen. In other studies underway we have extended our studies with the spe3 (delta)-fms1 (delta) mutants, using microarray techniques to study which systems are most affected by the addition of spermidine to polyamine-deficient S. cerevisiae mutants. We have found a dramatic induction of 73 genes, with an interesting concentration of genes involved in sulfur amino acid metabolism, transport, and plasma membrane function. Spermine supplementation had a much smaller effect, consistent with our other studies on the specificity in function of the different polyamines..